1. Field of the Invention
The present invention relates to a fluorescence detection apparatus that detects fluorescence from a fluorescent object. In particular, the present invention is applied to a fluorescence detection apparatus used for clinical tests such as DNA analysis.
2. Description of the Related Art
In an apparatus for imaging a fluorescence intensity of a marker of a target DNA bound with a probe of a DNA chip, a method is known in which fluorescent images are collectively captured with a long exposure because the fluorescence emitted from a marker is weak. Unfortunately, when the long exposure is performed, dark current may be generated in a sensor, causing a noise in an image. To avoid this, a cold charge-coupled device (CCD) has been used.
To remove the noise without a cold CCD, a method is known in which a noise image is generated while preventing light from entering an object after the object is captured with a long exposure, and the noise image is subtracted from the captured image of the object for its correction. For example, this is a method in which a noise image accumulated for a time equivalent to the exposure time is captured while a shutter is closed, and the noise image is subtracted from the captured image.
Also, there are known configurations that remove fixed pattern noise during a long exposure by using a dark image captured before the object is captured. Such configurations include: [1] a camera that previously acquires a plurality of dark images for a time shorter than an accumulation time calculated on the basis of a photometric value of an object, and obtains a dark image captured during the accumulation time, to reduce an acquisition time of the dark image (see Japanese Patent Laid-Open No. 2005-45552), and [2] a camera that captures dark images for an accumulation time calculated on the basis of a photometric value of an object before and after the actual capturing, to perform image correction by using both images (see Japanese Patent Laid-Open No. 2003-78820).
Also, an image converting and recording apparatus is known which measures the brightness of the outside through a photometric window, and calculates the accumulation time of a CCD (see Japanese Patent No. 03116397).
However, with these conventional examples, the method using the cold CCD is not practical because the cold CCD is extremely expensive, and hence, the apparatus may be expensive.
In contrast, an effective method is one, in which the light from the object is blocked, the noise image caused by an image pickup element or a peripheral circuit is captured and a fixed pattern noise image is generated, to correct the object image. The fixed pattern noise image may vary depending on the accumulation time of the image pickup element. Thus, the method captures the noise image for the accumulation time equivalent to that for capturing the object. The noise image for the accumulation time equivalent to that for capturing the object can be captured after the image of the object is captured. This may provide an accurate fixed pattern noise image. However, capturing the noise image after capturing the object requires time for correcting the fixed pattern noise, thereby increasing the testing time. This configuration is not practical when a large number of samples have to be measured.
Another issue to be considered is that the noise image captured while the light from the object is blocked contains a fixed pattern noise and a random noise. Since the random noise may vary depending on an image, the influence of the random noise should be removed and only the fixed pattern noise should be used for correction, to obtain a further accurate fluorescent image. To obtain this image, an effective method is one, in which a fixed pattern noise image is generated by capturing and averaging a plurality of dark images. However, capturing the plurality of dark images may require time. Further, if images of a plurality of DNA chips are captured, capturing a plurality of dark images for each DNA chip may require more time.
Therefore, the fixed pattern noise image has to be generated before the fluorescence is captured. It is, however, difficult to calculate the accumulation time in such an arrangement.
Japanese Patent Laid-Open No. 2005-45552 discloses a method in which the brightness of an object to be captured is measured through a taking lens. Prior to that image capture, the method sets two types of accumulation times, which are calculated on the basis of the measured light quantity and shorter than the exposure time for image-capturing. Then, non-exposure images previously captured during the accumulation times are used to correct the fixed pattern noise. However, when a dark image for a predetermined time is calculated using the dark images captured during the different accumulation times, the influence of the random noise may be enhanced. To remove the random noise, a larger number of dark images have to be taken.
In addition, the color of the fluorescent marker may fade, and the fluorescence intensity may deteriorate because of the irradiation of the exciting light. Thus, to measure accurately, light should not be emitted before the measurement. The brightness of the object cannot be previously measured through the taking lens, and thus, the accumulation time cannot be calculated.
In addition, as long as the exposure time is determined by using the brightness of the object, an image having a constant integration value of the light quantity is captured. It is not possible to obtain an image which has an absolute value of the fluorescent brightness for each probe.
Japanese Patent Laid-Open No. 2003-78820 is similar to the above in that the exposure amount is determined on the basis of the brightness of an object.
Also, with the method disclosed in Japanese Patent No. 03116397, which detects the brightness of the circumference using a built-in sensor, the accurate light quantity for illuminating the object (in this case, a silver-halide film) is not detected. Thus, it is difficult to obtain an accurate fluorescent image.
If the exciting light source excites the fluorescent marker with a constant brightness, a noise image and a fluorescent image can be captured during a predetermined accumulation time. However, such a laser source (in particular, a solid laser source with a constant output) is expensive, and the cost of a product with such a laser source may be expensive. On the other hand, the output of a low-cost laser source may vary due to the influence of temperature, heat-radiation environment, and the like, and may deteriorate with lighting time, thereby hardly providing stable illumination.